This invention relates to an improved synthesis of soluble phosphatides from phospholipids using phospholipase D enzyme as a catalyst, whereby high yields of high purity soluble phosphatides are obtained.
Phosphatides such as phosphatidyl glycerol are valuable and useful products used for making liposomes and lipid complexes.
Phosphatidyl glycerols and other phosphatides have been made heretofore by mixing an aqueous buffer solution containing calcium acetate, acetic acid and an enzyme, phospholipase D, and glycerol or other primary alcohol, with a phosphatidyl lipid, such as phosphatidyl choline, dissolved in a water immiscible organic solvent. In order to activate the enzyme, either a solvent such as ether has been used, or a surfactant has been added to emulsify the mixture of water insoluble and aqueous solutions.
Dimethyl ether, diethyl ether and other ethers, as has been disclosed by Redemann, PCT Application No. WO 89/01524 published Feb. 23, 1989, have been used to activate the enzyme, but these are known to be hazardous because of their flammability and their peroxide forming properties, which promote the auto-oxidation of the phosphatides. In addition, because of the very low density of ethers as compared to water, good mixing of the mixture of phases requires vigorous shaking, which can be difficult to scale up to commercial quantities.
Surfactants are useful also to activate the enzyme, but they are difficult to remove from the desired product. Thus, elaborate and expensive column chromatography separations are required to obtain a phosphatide of useful purity. Further, the presence of water in relatively large amounts results in hydrolysis and the concurrent production of phosphatidyl acid, which reduces the yield of the desired phosphatide product, and which also must be separated from the desired phosphatide.
Further, the enzyme requires an optimum pH range which necessitates the use of buffer solutions. The enzyme also requires a divalent cation such as calcium ion in the reaction mixture which produces the phosphatide as the calcium or other divalent cationic salt, which precipitates out of solution and therefore is difficult to solubilize. If acidification or ion exchange resin and neutralization are used to convert the calcium salts to their more soluble monovalent salts, very rapid hydrolysis occurs, with the concomitant precipitation of products of decomposition such as lysophosphatidyl glycerol or phosphatidyl acid, with the problems enumerated above.
In an attempt to improve the yields of phosphatides such as phosphatidyl glycerol, a process whereby a phosphatidyl lipid is reacted in an organic solvent with phospholipase D fixed on a carrier having hydrophobic groups has been disclosed. The solvent can be diethyl ether or an alkane which can dissolve phosphatidyl lipids such as phosphatidyl choline. The reaction is carried out at a temperature below the boiling point of the organic solvent, such as 15.degree. to 35.degree. C. However, yields of the desired phosphatide are low, on the order of 45%, and use of the ether solvents is inconvenient because they are highly flammable and dangerous solvents.
Thus, a method to produce phosphatides in a safe, simple manner in improved yield and in the form of a water soluble, monovalent, stable salt, has long been sought.
Previously, this transesterification reaction was usually done in a two phase system with ether in order to activate the reaction to a useful rate. However, the reaction was seldom quantitative as significant quantities of phosphatidic acid were also generated. In addition, due to the large difference in densities between the ether and the aqueous phase, large scale reactions gave limited yields due to insufficient mixing. Detergents may be used, but resulted in an increased difficulty in purifying the product.